影响牛胚胎干细胞分离克隆因素的研究

Influences on Isolation and Cloning of Embryonic Stem Cells From Bovine Primordial Germ Cells

采用与同源胎儿成纤维细胞共同培养及传统饲养层培养方式 ,以高糖DMEM ,添加 0 .1mM2 -巯基乙醇、犊牛血清、细胞因子为培养基 ,以 4～ 13周龄屠宰牛胎儿为实验材料 ,探讨影响牛原始生殖细胞分离克隆胚胎干细胞的相关因素。结果发现 :当犊牛血清为 15 %时效果最好 ;细胞因子添加与否对胚胎干细胞的分离及同源牛胎儿成纤维细胞的贴壁与生长影响并不显著 ,而在传代过程中有一定影响 ;以 0 .2 %胰酶 +0 .0 4 %EDTA为细胞消化液效果最佳 ;以同源胎儿成纤维细胞共培养的方式分离克隆牛胚胎干细胞 。

Primordial germ cells(PGCs) were isolated from 4～13 week old bovine fetus. Some of them were cultured on mitotically inactive feeder layer cells, the others and bovine embryonic fibroblast obtained from a conceptus of equivalent gestational age were cocultured. The culture system were composed of DMEM (high glucose) supplemented with NBS, 0. 1mM beta-mercaptoethanol, cytokines, The results attained were as follows:bovine ES cells in presence of 15% NBS was easiest to isolate and clone; The supplemented cytokines influence on isolation of ES cells and growth of homologous bovine fetus fibroblast wasn′t remarkable. Nevertheless, the influence was prominent in the process of cloning; the project of 0.2% trypsin +0.04% EDTA was relative gentle to cells and the capability of cloning was better,The manner of bovine ES cells and homologous fetus fibroblast coculture was dominant than the one of ES cells cultured depended on mitotically inactive feed layer.